The invention concerns a biochemical assay and a process for determining at least one target analyte in a biochemical sample, typically glycated and total haemoglobin in a blood sample. The invention also concerns an analytical test element on the basis of such a biochemical assay.
The glycation of haemoglobin is increased in the blood of diabetes patients. The increase depends on the concentration of glucose, free to move through the erythrocyte membrane and the period of incubation of the protein with glucose, via a non-enzymatic reaction. Hence, the determination of glycated haemoglobin (namely HbA1c) allows a retrospective estimate of the average glucose concentration and thus of the quality of the metabolic control of the diabetic patient. The disappearance of HbA1c from the blood depends on the lifetime of the erythrocytes (the average lifetime of these cells is about 120 days with a half-life of 60 days). HbA1c is defined as haemoglobin A that has been glycated by glucose with a slow but irreversible reaction on the N-terminal valine residues of the β chains. The HbA1c value is usually stated as a percentage of the total haemoglobin which requires a determination of the haemoglobin concentration from the same blood sample in addition to the HbA1c content.
In connection to this, it is already known to use test elements in order to provide a simple handling and rapid determination. A test element is generally understood as a carrier-bound (micro) system which enables sample preparation for an immediate analysis independent of a laboratory environment. Such test elements are usually intended to be single-use articles or disposables for near patient diagnostics in which all reagents that are necessary to carry out the test are provided on the carrier or component so that they can be used without requiring special handling.
In this context, U.S. Pat. No. 6,399,293 B1 discloses a teststrip-based system comprising a sample application zone, a reagent zone containing non-immobilized signal-generating molecules, a separation zone for separating the excess signal-generating molecules that are not bound to glycated haemoglobin and a detection zone. The separation occurs by means of a positively charged membrane binding only the excess of a negatively charged signal-generating ligand in the separation zone. The total haemoglobin including the labeled HbA1c will not be bound to the membrane and thus can be transported through the different zones within the sample liquid.
More generally, the use of test elements for different binding assays is well-known. For example, U.S. Pat. No. 4,094,647 describes a device that comprises a material capable of transporting a solution by capillary action. Different zones on the strip contain the reagents needed to perform the binding assay and to produce a detectable signal as the analyte is transported through the zones. The binding reaction occurs between an antigen and a complementary antibody. Many variations of the method have followed. However, despite all the activity in this field, methods have been developed always in the same direction involving the use of some immobilized reagent, mostly antibody, resulting in higher effort and costs for reagents and chemistry integration, and/or involving a chromatographic run, generally requiring the need for the analyte to go through several steps in space and time to meet the reaction and detection conditions.